Toxic materials may be introduced into the blood of humans by accidents, from disease states, from bacterial or viral infections, or from administration of substances for treatment of certain diseases (e.g. cancer therapy). Many of these toxic materials may do considerable damage to body tissues such as kidney, liver, lung and bone marrow, and may even be fatal. It is desirable to remove such materials from the blood as quickly as possible. Although the body has natural defense mechanisms to remove unwanted toxic materials, those methods can be ineffective in many examples. Thus, certain toxic materials are best removed from the blood in an extracorporeal device. An example of such a device is the kidney dialysis machine, where toxic materials build up in the blood due to a lack of kidney function. Other medical applications where an extracorporeal device can be used include: [1] removal of radioactive materials, [2] removal of toxic levels of metals, [3] removal of toxins produced from bacteria or viruses, [4] removal of toxic levels of drugs, and [5] removal of whole cells (e.g. cancerous cells, specific hematopoietic cells—e.g. B, T, or NK cells) or removal of bacteria and viruses.
In order for the extracorporeal device to function in toxin removal, it must have a chemical entity bound on it that has a high binding affinity with the toxic material that is to be removed from blood. Rather than binding that chemical entity directly to the column matrix in the extracorporeal device, it is preferentially bound through another binding pair of molecules. This arrangement of binding is used to make the toxin binding moiety more available in the blood and to make the device more generally applicable to a variety of toxic materials. A column matrix material is used that provides a high surface area while not restricting the flow of blood through it (Nilson, R. et. al. EPC 567 514). The column matrix has a protein (avidin or streptavidin) bound to it that has a high affinity for another molecule (e.g. biotin). That column is conditioned for use in a particular medical application by conjugation of a moiety that has a high affinity for the toxic material with two molecules at biotin such that attachment to the column matrix can be readily achieved. This conditioning reagent contains two biotin moieties rather than one as this configuration provides a higher degree of stability to the column matrix.
Although, tumor-specific immunoconjugates are selectively bound to tumor cells, an initial high concentration of the cell-toxic immunoconjugate in the blood circulation is necessary to reach a sufficient high concentration of the target tissue in a patient. While required for optimal therapy of the cancer, the high concentration of cytotoxic material in the blood and other non-tumor tissues, in most cases leads to tissue damage and/or lesion formation in sensitive and vital tissues like the bone marrow. Although, bone marrow rescue is sometimes used to circumvent these potentially lethal effects, such transplantation is both extremely costly and possesses a high risk for the patient. Even in cases where the bone marrow transplantation is effective, other sensitive organs like the, liver, kidney, spleen, lung etc. can be irreparably damaged. The most effective method for preventing tissue and bone marrow damage from toxic materials in blood is to dramatically decrease the amount of that toxic material in the blood. Of course, this must be accomplished in a manner that retains the therapeutic level of toxic material in the tissue being treated (e.g. tumor).
Radiolabeled antibodies have been under investigation for therapy of cancer for several decades. Administration of radiolabeled antibodies introduces a toxic material into blood. Various methods have been proposed to rapidly clear radiolabeled antibodies from blood circulation after the tumor has accumulated a sufficient quantity of immunoconjugate to obtain a diagnosis or therapy. Some of the methods employed involve enhancement of the bodies own clearing mechanism through the formation of immune complexes. Enhanced blood clearance of radiolabeled antibodies can be obtained by using molecules that bind it, such as other monoclonal antibodies (Klibanov et. al., J. Nucl. Med. 29, 1951-1956, 1988; Marshall et al. Br. J. Cancer 69, 502-507, 1994; Sharkey et al. Bioconjugate Chem. 8, 595-904, 1997), avidin/streptavidin (Sinitsyn et al., J. Nucl. Med. 30, 66-69, 1989; Marshall et. al., Br. J. Cancer, 71, 18-24, 1995), or glycosyl containing compounds which are removed by receptors on liver cells (Ashwell and Morell, Adv. Enzymol. 41, 99-128, 1974). Still other methods involve removing the circulating immunoconjugates through extracorporeal methods (see review article by Schriber, G. J. & Kerr, D. E., Current Medical Chemistry, 1995, Vol. 2, pp 615-529).
The extracorporeal techniques used to clear a medical agent from blood circulation are particularly attractive because the toxic material is rapidly removed from the body. Application of these methods in the context of immunotherapy have been previously described (Henry C A, 1991, Vol. 18, pp. 565; Hofheinze D et al., Proc. Am. Assoc. Cancer. Res. 1987 Vol. 28, pp 391; Lear J K, et al. Radiology 1991, Vol. 179, pp. 509-512; Johnson T. K. et. al. Antibody Immunoconj. Radiopharm. 1991, Vol. 4, pp. 509; Dienhart D. G., et al. Antibody Immunoconj. Radiopharm. 1991, Vol. 7, pp. 225; DeNardo G. L. et al. J. Nucl. Med. 1993, Vol. 34, pp 1020-1027; DeNardo S. J. et. al. J. Nucl. Med. 1992, Vol. 33, pp. 862-863; DeNardo G. L. J. Nucl. Med. 1992, Vol. 33, pp. 863-864; and U.S. Pat. No. 5,474,772; Australian Patent 638061, EPO; and EPO 90 914303.4 of Maddock.
To make the blood clearance more efficient and to enable processing of whole blood, rather than blood plasma as the above methods refer to, the medical agents (e.g. tumor specific monoclonal antibody carrying cell killing agents or radionuclides for tumor localization) have been biotinylated and cleared by an avidin based adsorbent on a column matrix. A number of publications provide data showing that this technique is both efficient and practical for the clearance of biotinylated and radionuclide labeled tumor specific antibodies (Norrgren K, et. al. Antibody Immunoconj. Radiopharm. 1991, Vol. 4, pp 54; Norrgren K, et. al. J. Nucl. Med. 1993, Vol. 34, pp. 448-454; Garkavij M, et. al. Acta Oncologica 1996, Vol. 53, pp. 309-312; Garkavij M, et. al. J. Nucl. Med. 1997, Vol. 38, pp. 895-901. These techniques are also described in U.S. patent application Ser. No. 08/090,047; EPC 567 514 and Ser. No. 08/434,889).
Apart from the prolonged circulation time leading to undesired exposure of toxic immunoconjugate to healthy tissue, inadequate tumor tissue penetration and non-specific organ retention and metabolism contribute to a low therapeutic index ratio. Due to these problems, multi-step antibody-based radionuclide delivery approaches have been extensively investigated. The basic concept involves first the injection of a lesion-specific targeting moiety which apart from binding specifically to the lesion also has the feature of binding to a subsequently injected radioactive diagnostic agent or a therapeutic agent. By separating these two events one can allow the slow tissue penetrating non-radioactive/non-cytotoxic antibody sufficient time to accumulate in the tumor mass, while the agent carrying the radionuclide/cytotoxin could be selected for more rapid tissue penetration. However, a prerequisite is that the former (and preferably also the later) can be cleared rapidly from the blood circulation.
Most of these multi-step approaches utilize binding pairs of avidin/streptavidin and biotin. Avidin is a 67 kDa glycoprotein found in egg whites and tissue of birds and amphibia. It consists of 4 non-covalently bound subunits. Each subunit is capable of binding one biotin molecule. Avidin has a high isoelectric point (pI>10), due to its 36 lysine amino acid residues, which results in non-specific binding to cellular membranes. Streptavidin (SAv), produced in Streptomyces avidinii, is a close relative of avidin. It shares high affinity to biotin, but differs in amino acid content as well as net charge (pI 6.5) and is not glycosylated. Due to lack of sugar groups, SAv has a slightly lower molecular weight of 60 kDa and the in vivo pharmacokinetics and biodistribution differs markedly from avidin. Whereas intravenous injection of radiolabelled avidin clears rapidly from the blood and accumulates extensively in the liver, radiolabeled SAv exhibits a much longer circulation time, and has lower organ accumulation (Pima M V et al. Nucl. Med. Comm. 1988 Vol. 9, 931-941; Schechter B et al., Eur. J. Biochem. 1990, Vol. 189, 327-321; Rosebrough S F, Nucl. Med. Biol. 1993, Vol. 20, 663-668).
The other part of the binding pair, biotin, is a vitamin and a member of the B-complex, which is essential for amino acid and odd-chain fatty acid synthesis. Biotin in found preferentially intracellular, usually bound to an enzyme and acts as a co-factor during carboxylation reactions. Biotin is often present as a lysine-biotin adduct (biocytin), in food and during metabolic protein turnover. The linkage between lysine and biotin is cleaved by a plasma enzyme, biotinidase.
To improve the imaging in patients with carcinoma of the lung, Kalofonos et. al. used a two-step SAv-MAb/111In DTPA-biotin approach (Kalofonos H P et al., J. Nucl. Med. 1990, Vol. 31, 1791-1796). Van Osdol et. Al. And Sung et. al. have developed a mathematical model of two-step imaging, and treatment protocols using SAv-MAb and radiolabelled biotin chelates. Taken into account the in vivo parameters of both the targeting SAv-MAb moiety and the radiolabelled biotin imaging agent, they predicted the following:
1) The large molecular weight of SAv-MAb will reduce the amount of MAb that will localize in the tumor and the binding homogeneity in the tumor.
2) Radiolabelled biotin will diffuse rapidly into the tumor, but due to the high affinity to peripheral tumor-bound SAv-Mab, will not penetrate deeply into the nodule if too low dose is given.
3) Compared to directly labeled MAbs, the two-step SAv-MAb/radiolabelled biotin protocol permits imaging sooner after radioactive injection and produced higher tumor/blood ratios.
4) That tumor/blood ratios at 24 hrs are >2 times higher that with the use of directly labeled MAbs.
In their simulation, a high percentage of the radio activity is bound to circulating SAv-MAb and that the addition of clearing agent before radiolabelled biotin was injected would enhance the tumor blood ratio.
A two-step approach using biotinylated MAbs and radiolabelled SAv has also been utilized in animal models as well as in patients (Paganelli G. et. al. Eur. J. Nucl. Med. 1992, Vol. 19, 322-329; Khawli L A et al. Abs. Immunoconj. Radiopharm. 1993 Vol. 6, 13-27; Kassis A I. et. al. J. Nucl. Med. 1996 Vol. 35, 1358-1365). In this method, both the targeting and the imaging agents are of large molecular weight and clear slowly from the blood. The whole procedure takes many days to complete and with metabolism, radioactivity accumulates in organs and is slowly eliminated from the body. Nevertheless, these studies showed that biotin/SAv binding was accomplished in vivo and yielded positive images and enhanced tumor activity compared to directly labeled MAbs.
A three-step procedure consisting of biotinylated MAb, avidin and then followed by 111In-DTPA-biotin has also been tried (Paganelli G et al. Canc. Res. 1991 Vol. 51, 5960-5966; Dosio F. et. al. J. Nucl. Biol. Med. 1993 Vol. 37, 228-232). This procedure required 1-3 days between injections to allow for tumor accumulation and blood clearance. As a whole, all these studies have shown the feasibility of immunological approaches utilizing the SAv/biotin system in vivo. However, circulating levels of the high molecular weight targeting agents were problematic due to their prolonged circulation and non-specific organ accumulation.
An alternative pretargeting approach uses three separate injections of three components: [1] SAv-Mab, [2] a clearing agent, and [3] a radiolabelled biotin derivative containing the radiometal chelation moiety DOTA has been thoroughly investigated (Axworthy D B et al. J. Immunother. 1994, Vol. 16, 158). A covalent conjugate of tumor-specific MAb and SAv is injected and is allowed to accumulate at tumor sites. After sufficient tumor uptake (24-48 hrs) a biotin clearing agent is administered in order to clear the blood from the conjugate through the liver. Finally, the radiolabelled biotin-DOTA derivative is injected. The clearing agent used in this context is typically a biotinylated protein to which galactose residues have been conjugated. The galactose receptors resides on hepatocytes and exhibit a high affinity and specificity for macromolecules with exposed terminal galactose residues. The hepatic uptake correlate with the amount of galactose residues bound to SAv (Rosebrough S F, J. Nucl. Med. 1996, Vol. 37, 344-350).
In all these concepts there is bound to be a conflict between initial concentration of the targeting molecule and its ability to penetrate deep into the tumor on one hand, and a rapid and complete clearance from the blood prior to administration of the radioactive/cytotoxic agent, on the other. In principle, the same condition applies for the radioactive/cytotoxic agent. A sufficiently high initial blood concentration is essential to reach and saturate the targeting molecule. At the same time this toxic agent must not reside in the blood circulation and exposing sensitive tissues like the bone marrow. Even if the toxic agent is cleared fairly rapidly through the body, organs like the kidney and the urinary track will normally receive an accumulated toxic dose equally or higher to that received by the tumor tissue.
There is clearly a need for optimizing these and other therapy protocol conditions, particularly it the approaches are going to be adequate for the treatment of solid tumors. It is vital that such concepts are to a large extent generic, in so far that as many as possible of the parameters are independent on the type and localization of the disease and as much as possible independent on the pharmacokinetic parameters and rate of metabolisms of the individual patient.